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Back to Journal »International Journal of Nanomedicine» Volume 12

Authors: Yin Jie, Xiang C, Wang Ping, Yin Y, Hou Y

Published on April 10, 2017 2017 Volume: 12 pages 2923-2931

DOI https://doi.org/10.2147/IJN.S131167

Single anonymous peer review

Editor who approved for publication: Dr. Linlin Sun

Juntao Yin,1,*崔玉祥,1,* Peiqing Wang,1 Yuyun Yin,2 Yantao Hou3 1 Department of Pharmacy, Huaihe Hospital Affiliated to Henan University, Kaifeng, 2 Department of Physical and Chemical Analysis, Henan Food and Drug Inspection Institute, Zhengzhou, 3 Henan Applied Technology Occupation Faculty of Pharmaceutical Engineering, Kaifeng, People’s Republic of China* The above authors have contributed equally to this study. Abstract: Baicalein (BCL) has high pharmacological activity, but its solubility and stability in the intestinal tract is low. This study aims to explore the potential of nanoemulsions (NEs) composed of hemp oil and less surfactants in improving the oral bioavailability of BCL. BCL-loaded NEs (BCL-NEs) are prepared by high-pressure homogenization technology to reduce the amount of surfactants.​​​ The characteristics of BCL-NEs are particle size, encapsulation efficiency (EE), in vitro drug release and morphology. The bioavailability of Sprague-Dawley rats was studied after oral administration of BCL suspension, BCL conventional emulsion, and BCL-NE. The obtained NE has a particle size of about 90 nm and an EE of 99.31%. BCL-NEs significantly improved the oral bioavailability of BCL, compared with suspensions and conventional emulsions, as high as 524.7% and 242.1%, respectively. BCL-NEs show excellent intestinal permeability and trans-cell transport ability. The cytotoxicity of BCL-NEs is proven to be low and can be used for oral administration. Our research results show that this new type of NE and preparation process provide a promising alternative to current formulation technology and is suitable for oral administration of drugs with bioavailability issues. Keywords: baicalein, nanoemulsion, biocompatibility, high pressure homogenization, sesame oil, bioavailability

Baicalein (BCL) is a flavonoid compound rich in the root of Scutellaria baicalensis Georgi. It is reported to have a variety of pharmacological activities, such as anti-cancer, antioxidant, anti-allergic, anti-viral and anti-inflammatory activities. 1 BCL is also one of the active ingredients of Scutellaria baicalensis Georgi Sho-Saiko-To, a Japanese herbal supplement believed to improve liver health. Very insoluble in acidic media, soluble in alkaline media, but unstable. Poor solubility in gastric juice and instability in intestinal juice greatly limit its oral bioavailability and clinical efficacy. In order to improve the oral absorption of BCL, many new formulation methods have been explored, including solid dispersions, 2 cyclodextrin inclusion complexes, 2 nanocrystals, 3 self-microemulsification, 1 phospholipid complex, 4 and liposomes. 5 However, these methods still have some shortcomings of being used as carriers for oral administration due to insufficient drug protection, poor biocompatibility, and large particle size. There is an urgent need to develop a low-toxic and high-efficiency BCL oral drug delivery system.

Nanoemulsions (NEs) are oil-in-water or water-in-oil droplets emulsified by surfactants, co-surfactants, oil and water in an appropriate ratio. 6 Generally, due to the use of a large amount of surfactants (wt, ~20% oil phase). The smaller particle size (10-100 nm) gives NEs excellent drug delivery efficacy, although the high surfactant content increases the safety risk of the delivery system. The formulation characteristics of NEs depend to a large extent on the formulation ingredients and preparation technology. 6 Appropriate technology and carrier materials can jointly reduce the amount of surfactant used, thereby reducing the toxicity of NEs. In addition to spontaneous formation, NEs can also be manufactured by applying strong mechanical power, which can greatly reduce the amount of surfactants.​​​ High pressure homogenization (HPH) technology is widely used to produce submicron lipid emulsions. 7 This should be an effective way to use HPH to prepare NEs to minimize the surfactant content in NEs. Hemp oil extracted from hemp seeds has become an important source of edible oil for human nutrition. In addition to some health effects, 8,9 due to its high water solubility, it is easier to emulsify by emulsifiers than other commonly used oils. This will help to further reduce the surfactant in NE. However, the cannabis oil-based NEs prepared by HPH technology has not been reported and applied to the oral administration of BCL.

In this study, NEs composed of hemp oil and reduced surfactants were developed to improve the intestinal stability and permeability of BCL, thereby increasing the oral bioavailability (as shown in Scheme 1). We prepared BCL-loaded NEs (BCL-NEs) using HPH technology, and characterized them by particle size, retention efficiency (EE), morphology, and in vitro release. The ability of NEs to increase the bioavailability of BCL was studied in Sprague-Dawley (SD) rats and compared with suspensions and conventional emulsions. In order to evaluate the performance of the preparation, the intestinal permeability, transcellular transport and cytotoxicity of BCL-NE were examined.

Scheme 1 Illustration of the preparation of NEs and oral administration of BCL. Abbreviations: BCL, baicalein; HPH, high pressure homogenization; NEs, nanoemulsion; PM, poly(ethylene glycol) monooleate.

BCL was purchased from Zhengzhou Agricultural Technology Co., Ltd. (Zhengzhou, People's Republic of China). Hemp oil was purchased from Impressions of Life Experience Industry Co, Ltd (Bama, People's Republic of China). Poly(ethylene glycol) monooleate (PM; Mw~1,400) was purchased from Aoke Chemicals (Shanghai, People's Republic of China). Sodium oleate, Hoechst 33258 and 3,3'-Dioctadecanocyanine perchlorate (DiO) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Deionized water was prepared by Milli-Q water purifier (Molsheim, Millipore, France). High performance liquid chromatography (HPLC) grade methanol was supplied by Merck (Darmstadt, Germany). All other chemicals are of analytical grade and used as is.

Add a slight excess of BCL to 2 mL of each test carrier (soybean oil, medium chain triglycerides [MCT], Capryol™ 90, hemp oil, PM, and hemp oil/PM), and stir the mixture for 48 hours. At 25°C and 800 rpm. After equilibration, the mixture was centrifuged at 12,000 rpm for 10 minutes. The BCL in the supernatant was quantified by HPLC. The measurement was performed on an Agilent 1100 series HPLC system (Santa Clara, California, USA). The sample was eluted through a Hypersil™ C18 column (5 μm, 4.6×200 mm; Elite, Dalian, China, People’s Republic of China) at 35°C with an injection volume of 20 μL. Collect the chromatographic signal at 276 nm. The mobile phase is composed of 46% methanol and 54% phosphoric acid solution (0.1%), with a flow rate of 1.0 mL/min.

BCL-NEs are prepared through the emulsification/HPH process. In short, mix BCL, sesame oil, PM and appropriate amount of ethanol and heat (50°C) to melt, and dissolve sodium oleate in deionized water. Then, the water phase was transferred to the oil phase, and then a high-speed disperser (T25; IKA, Staufen, Germany) was used to perform high shear at 8,000 rpm for 5 minutes to produce a coarse emulsion. The obtained coarse emulsion was further homogenized with a Microfluidizer (Nano DeBEE, Westwood, MA, USA) to produce the final NE. The factors affecting NE properties were screened, including drug/excipient, surfactant/oil ratio and homogenization conditions. The surfactant is composed of PM and sodium oleate in a fixed ratio of 1:1. BCL suspensions and conventional emulsions were prepared by grinding the drug with sodium carboxymethyl cellulose (0.5%) or the excipients used in BCL-NE as a control formulation.

The particle size of BCL-NE is determined by Zetasizer Nano ZS (Malvern, Worcestershire, UK) at 25°C. In order to measure the particle size, BCL-NEs were appropriately diluted and placed in a disposable cuvette. After equilibrating for 120 s, laser diffraction was performed on the sample, and particle size analysis based on dynamic light scattering was performed.

Check the morphology of BCL-NEs with a transmission electron microscope (TEM). Dilute BCL-NE to 25 times, drop it on the carbon-coated copper net, and then air dry and attach. Then, the fixed particles were forwarded to TEM (Tecnai 10; Philips, Amsterdam, Netherlands) for observation. Morphological micrographs were collected at an acceleration voltage of 100 kV.

The EE percentage of BCL-NEs is determined by separating free BCL from the emulsion system. In short, freshly prepared BCL-NE is centrifuged in a centrifugal filter device (Amicon® Ultra-0.5, MWCO 10K; Millipore) to remove uncaptured BCL. The BCL in the filtrate was determined by the aforementioned HPLC. The EE of BCL-NEs is calculated according to the following equation: EE (%) = (1− Mfre/Mtot) × 100%, where Mfre and Mtot represent the amount of free and total BCL in the system, respectively.

The dialysis bag method was used to study the release of BCL in BCL-NEs. 10 In short, put an aliquot of BCL-NEs equivalent to 25 mg BCL into a dialysis bag, and then place 900 mL of water, 0.1 M HCl solution or phosphate-buffered saline (PBS), pH 6.8 , Which introduces 0.25% (w/v) sodium lauryl sulfate as a solubilizer. At 0.5, 1, 2, 4, 8 and 12 h, take out 5 mL of the solution and immediately add the same volume of fresh medium. Subsequently, the release solution was analyzed by HPLC for BCL quantification, and the cumulative release percentage of BCL in BCL-NEs was calculated based on the ratio of the released drug to the total drug.

SD rats (250±20g) were fasted overnight before administration, but could drink freely. The rats were randomly divided into three groups with six rats in each group. The first group was given BCL suspension, while the second and third groups were given BCL conventional emulsion and BCL-NE, respectively. These three preparations were administered to rats by gavage at a dose of 25 mg/kg. At 0.5, 1, 2, 4, 6, 8 and 12 hours after administration, blood was sampled into the heparinized tube via the jugular vein. The blood sample was centrifuged at 4,500 rpm for 5 minutes to prepare plasma. All animal experiments were carried out in accordance with the protocol issued by the experimental animal ethics committee of Huaihai Hospital affiliated to Henan University, and all animal experiments were reviewed and approved by the ethics committee.

In order to quantify the plasma BCL and its glucuronic acid metabolite balokarine, a deproteinization procedure was performed by adding four aliquots of methanol to aliquots of plasma. After several vortexing and sonication, the sample was centrifuged at 10,000 rpm for 10 minutes to separate the supernatant. Then, the supernatant was evaporated to dryness at 37°C for 2 hours under vacuum using Concentrator plus (Eppendorf, Hamburg, Germany). Redissolve the residue in 100 mL of 50% acetonitrile aqueous solution. After centrifugation, the resulting solution was subjected to ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-qTOF/MS) analysis (Xevo G2 QTof; Waters, Milford, Connecticut, USA). Reference documents for instrument configuration and parameter settings. 11 Quantify the entire BCL in the blood based on the ion chromatogram extracted using MassLynx. The PKSolver 2.0 program was used to process the pharmacokinetic parameters.

In order to evaluate the intestinal permeability of BCL-NEs, in situ single intestinal perfusion was performed according to the reported procedure. Twelve SD rats were fasted for 12 hours before the experiment. The rats were then unconscious by injecting 20% ​​urethane. Expose the intestines by making a midline incision in the abdomen. The segments of the duodenum, jejunum, and ileum were identified and cannulated with silicone tubing. The intestinal contents were washed thoroughly with Krebs Ringer's buffer, pH 7.4 (Seebio Biotech, Inc, Shanghai, People's Republic of China). Prepare perfusion samples by diluting BCL (dissolved in ethanol) or BCL-NE into Krebs Ringer buffer to obtain a BCL concentration of 25 μg/mL. After 30 minutes of pre-perfusion, the perfusate was collected every 15 minutes until 120 minutes. In the sham operation group, the water flux was calibrated by weight. The effective permeability coefficient (Peff) is calculated according to the following formula:

Where Q represents the flow rate (0.2 mL/min), r and L are the radius and length (cm) of each intestinal segment, and Cin and Cout represent the inlet and outlet concentrations of BCL, respectively.

Caco-2 cells were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, People's Republic of China), from the American Type Culture Collection (ATCC), and used to study the cell uptake and internalization of BCL-NEs, as well as the culture according to the reported protocol13. The cultured cells were washed twice with pH 7.4 PBS, and seeded in a six-well plate at a density of 1×106 cells/well. When the cell fusion degree is close to 80%~90%, it is used for cell uptake experiments. The BCL solution and BCL-NE diluted with medium to 10 μg/mL were introduced into the wells containing Caco-2 cells. At 0.5, 1, 2, and 4 hours, remove the culture medium and wash the cells with cold PBS. Then, the treated cells were lysed using radioimmunoprecipitation assay lysis buffer (0.1% phenylmethanesulfonyl fluoride) (Sigma-Aldrich). The supernatant was obtained by centrifugation, and the protein content was quantified using the BCA protein determination kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, People’s Republic of China). The concentration of BCL in the supernatant was determined by the aforementioned UPLC-qTOF/MS and corrected by the cell protein level of each well.

In order to observe the cell internalization of BCL-NE, DiO-labeled BCL-NE was prepared by loading DiO into BCL-NE during preparation. Caco-2 cells were incubated with DiO-labeled BCL-NE at 37°C for 0.5 hours. The medium was removed and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde, and after staining with Hoechst 33258, the confocal laser scanning microscope (CLSM) was continued.

Check BCL by evaluating the effect of Caco-2 cell viability based on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) -Cytotoxicity of NEs) determination. Caco-2 cells were cultured for 48 hours and washed three times with PBS. Then, different levels of BCL-NEs were added to the cells and incubated at 37°C for 24 hours. After that, MTT solution (20 μL, 5 mg/mL; Sigma-Aldrich) was introduced into each well and incubated for another 4 hours. In order to dissolve the obtained formazan, 200 μL of dimethyl sulfoxide was then added. Measure the UV absorbance of each well at 570 nm. Use the following formula to calculate cell viability: cell viability (%) = (Atri/Acon) × 100%, where Atri and Acon represent the absorbance of live cells treated with BCL-NEs and blank medium, respectively.

The solubility of BCL in various vehicles

The main purpose of developing NE is to dissolve drugs that are insoluble in water. The solubility of the drug in the oil used for NE determines the solubilization ability and stability of the formulation. Table 1 shows the solubility of BCL in various carriers. Among the four tested oils, hemp oil has the highest solubility for BCL, and the solubility of BCL is as high as 8.592 mg/g. Generally speaking, the solubility of poorly water-soluble drugs is long-chain oil<medium-chain oil<short-chain oil. 14 Soybean oil is a representative of long-chain oil, and Capryol™ 90 is a short-chain oil. Oil. Interestingly, BCL exhibits excellent solubility in hemp oil, which is much higher than Capryol™ 90. This may be due to the hydrophilic nature of hemp oil. Sesame oil is known as water-based edible oil in China, and it is very miscible with water. PM is an amphiphilic material containing polyethylene glycol moiety. BCL is a polyphenolic compound (Figure 1), with insufficient hydrophilic and lipophilic balance, resulting in poor solubility in water and oil. Therefore, PM makes BCL easier to dissolve in it by virtue of its excellent solubilizing ability. The combined use of hemp oil and PM further increases the solubility of BCL. Our findings indicate that hemp oil and its combination with PM are more suitable for preparing NE for loading BCL.

Table 1 Solubility of baicalein in various excipients Note: solubility, milligrams of baicalein in 1 g of excipient; Capryol™ 90, propylene glycol monocaprylate. Data are expressed as mean ± StD (n=3). Abbreviations: MCT, medium chain triglycerides; PM, poly(ethylene glycol) monooleate; StD, standard deviation.

Figure 1 The chemical structure of baicalein.

Preparation and characterization of BCL-NEs

Generally, NEs can be formed by self-emulsification by stirring or vortexing in the presence of several emulsifiers. For example, in order to obtain a good econazole nitrate self-emulsifying drug delivery system, 60% (w/w) Cremophor® RH 40 and a high proportion of co-surfactant (Transcutol® HP) are used in the formulation. 15​​ However, the use of large amounts of emulsifiers or surfactants will produce high physiological toxicity. In order to minimize the use of surfactants and realize nano-scale emulsions, HPH is used in the processing of BCL-NE to reduce particle size by providing strong mechanical energy. This preparation technology is similar to the technology used in the preparation of solid lipid nanoparticles or nanostructured lipid carriers. 16,17 The particle size of the nanocarrier affects the intestinal absorption of the payload. In preliminary experiments, we found that the factors affecting the particle size of BCL-NEs mainly include drug/excipients, surfactant/oil ratio, and homogenization conditions. The influence of formula variables on particle size is shown in Figure 2. The drug/oil ratio has a significant effect on the particle size of BCL-NEs. Due to the impaired emulsification of the internal oil phase, high drug participation results in larger emulsion droplets. On the contrary, a high proportion of surfactants has a positive effect on particle size reduction. It is worth noting that a formulation that uses less surfactant (relative to 10% of oil) can still produce NE <100 nm. Under homogenization conditions, the particle size of BCL-NEs decreases as the homogenization period increases at 20,000 psi. But in terms of particle size, there is no significant difference between 8 and 12 cycles.

Figure 2 The effect of the ratio of drug/excipient (A) and surfactant/oil (B) and the homogenization cycle (C) of 20,000 psi on the particle size of BCL-NEs (mean ± standard deviation, n=3) , One of the factors is set as an independent variable, and the other two factors remain unchanged. *P<0.05; **P<0.01. Abbreviations: BCL, baicalein; NEs, nanoemulsion; StD, standard deviation.

Taking into account the advantages of smaller particle size and fewer surfactants during oral administration, the final formulation is 40 mg BCL, 1,000 mg cannabis oil, 50 mg PM, and 50 mg sodium oleate, emulsified with 20 mL of water. The obtained BCL-NE has a particle size of 90.6 nm and a polydispersity index of 0.225 (Figure 3A). The zeta potential was determined to be -45.2 mV, indicating that they are stable as a colloidal dispersion system. BCL-NEs showed a high EE as high as 99.31%. The appearance of BCL-NEs is milky white, and blue light after dilution (Figure 3B). BCL-NEs exhibited a nearly spherical morphology, as observed by TEM (Figure 3C). The particle size judged from the scale bar (200 nm) is in good agreement with the hydrodynamic size measured based on dynamic light scattering.

Figure 3 Physical characteristics of BCL-NE: (A) size distribution, (B) appearance and (C) TEM micrograph. Abbreviations: BCL, baicalein; NEs, nanoemulsion; TEM, transmission electron microscope.

The release curves of BCL from NEs in various media are shown in Figure 4. BCL-NEs showed extremely slow drug release in water, 0.1 M HCl and PBS, pH 6.8. The release amount increased over time in the three different media, indicating that BCL can be continuously released from BCL-NEs. However, due to the poor solubility of BCL, the release amount is quite low, and the solubility in the three media is <7% within 12 hours. Note that the release of BCL-NE in 0.1 M HCl is slightly different in water and PBS (pH 6.8). In 0.1 M HCl, BCL-NEs showed lower drug release, which may be related to the poor solubility of BCL in acidic media. In general, the release of BCL from BCL-NEs is quite limited, and most of the BCL remains in NEs. The publishing function enables BCL to be transmitted through NE vehicles instead of free form. Other research groups have also investigated the insignificant drug release of NE. 18,19

Figure 4 In water, 0.1 M HCl and PBS, pH 6.8 (n=3, average ± StD), over time, the release curve of BCL in nanoemulsion. Abbreviations: BCL, baicalein; PBS, phosphate buffered saline; StD, standard deviation.

The pharmacokinetic curves of BCL after oral administration of different dosage forms are shown in Figure 5, and the main pharmacokinetic parameters are listed in Table 2. Suspension caused poor absorption rate and degree of BCL. The maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-t) are only 1.43 μg/mL and 7.96 μg h/mL, respectively. Compared with BCL suspension formulations, conventional emulsions promote the oral absorption of BCL to a certain extent, and significantly improve the blood concentration. This finding suggests that lipid-based formulations can increase the oral bioavailability of BCL. However, in the case of BCL-NEs, the oral absorption of BCL is significantly enhanced. BCL-NEs produced higher blood BCL concentrations at each time point. Cmax and AUC0–t are as high as 10.96 μg/mL and 41.77 μg h/mL, respectively. The oral bioavailability of BCL-NEs is 524.7% and 242.1% of suspension and conventional emulsion, respectively. In terms of time to maximum plasma concentration (Tmax), the two emulsions are similar (~2 h), but the formulation of the suspension is different. In addition, the terminal half-life (T1/2) of the three preparations are also significantly different. These results indicate that there are differences in the absorption rate of BCL formulated in different ways. In contrast, NEs have greater advantages in enhancing the oral absorption of BCL.

Figure 5 The pharmacokinetic characteristics of BCL after oral administration of BCL suspension, BCL conventional emulsion and BCL-NEs in Sprague-Dawley rats (n=6, mean ± StD). Abbreviations: BCL, baicalein; NEs, nanoemulsion; StD, standard deviation.

Table 2 The pharmacokinetic parameters of BCL after oral administration of BCL suspension, BCL emulsion and BCL-NEs in rats. Note: AUC, area under the blood BCL concentration-time curve; comparison of RBA and BCL suspension. Data are expressed as mean ± StD (n=6). *P<0.01, which is significantly different from BCL emulsion and/or BCL suspension. "-" means not applicable. Abbreviations: BCL, baicalein; RBA, relative bioavailability; NEs, nanoemulsion; StD, standard deviation.

NEs are nano-sized colloidal particles, which represent one of the most advanced nanoparticle systems for oral administration. 20 NEs are thermodynamically and kinetically stable, so flocculation, aggregation, creaming and coalescence rarely occur. The high surface area can increase the absorption rate and reduce the absorption variability, thereby increasing the bioavailability of the drug. In addition, because they are encapsulated in the internal oil phase, they can protect the payload from degradation and metabolism, 21 similar to a micellar system. 22 In terms of stabilizing unstable drugs, NEs is superior to solid dispersions and cyclodextrin inclusion compounds. However, the main disadvantage of NEs involves the high concentration of surfactants/co-surfactants required for stabilization. Compared with the self-microemulsifying drug delivery system 1, the NEs developed by us contain less surfactants and co-surfactants with the help of HPH during preparation. The new NEs with hemp oil as the carrier have higher oral administration efficacy and are more suitable for oral administration of BCL.

Drug permeability can be assessed by Caco-2 cell monolayer model or in situ single-channel intestinal perfusion. 23,24 In this study, in situ single-channel intestinal perfusion was used to measure Peff. The Peff values ​​of free BCL and encapsulated BCL in NEs are listed in Table 3. BCL itself has poor permeability in each intestinal segment, Peff<5×10-6 cm/s. It seems that BCL has a higher permeability in the lower part of the small intestine. After being encapsulated in NEs, the Peff of BCL in the main absorption intestinal area was significantly increased, especially the ileum. In contrast, the ileum has a larger absorption area and transport activity. We hypothesize that the increased surface area and particle-related membrane transport should be the reason for the increased permeability. 25 The in situ single-pass intestinal perfusion experiment proved the positive effect of NEs in enhancing the oral absorption of BCL.

Table 3 Effective intestinal permeability (Peff) of free BCL and encapsulated BCL in NE measured by in situ single-pass intestinal perfusion. Note: Paired t-test. Data are expressed as mean ± StD (n=8). *P<0.01, which is significantly different from free BCL. Abbreviations: BCL, baicalein; NEs, nanoemulsion; StD, standard deviation.

Figure 6A shows the cellular uptake of free BCL and BCL-NE with incubation time. There is a significant difference in cellular uptake between free BCL and encapsulated BCL. At the first time point (0.5 hours), free BCL was taken up by Caco-2 cells, a little faster than BCL-NEs. This may be related to the high concentration of BCL around the cells due to complete exposure. However, after that, NEs accelerated the cellular uptake rate of BCL compared to free BCL. At 4 hours, the total intake of BCL-NEs was 1.86 times that of free BCL. BCL-NEs lead to higher BCL uptake, suggesting that they have the potential to promote the translocation of BCL across cells.

Figure 6 The cellular uptake of free BCL and BCL-NEs is determined by the intracellular drug level (A) (mean ± StD, n=3) and the cellular internalization of BCL-NEs assessed by CLSM imaging (B). *P<0.05; **P<0.01. Abbreviations: BCL, baicalein; CLSM, confocal laser scanning microscope; NEs, nanoemulsion; StD, standard deviation.

The promoting effect of BCL-NEs on BCL transport can also be inferred from the internalization of BCL-NEs. Strong cell internalization occurred on BCL-NEs (Figure 6B). A large amount of NE-related fluorescence is distributed in the cytoplasm, and even internalized into the nucleus. The enhancement of permeability through NE has been studied in other tested drugs. 26 NE's excellent affinity and permeability for intestinal cells make them eligible for oral malabsorption drugs.

For the application of NEs, the safety of the preparation is of great concern. The toxicity of NEs generally comes from the use of large amounts of surfactants and co-surfactants. Figure 7 shows the viability of Caco-2 cells after treatment with BCL-NEs. Under different drug concentrations, no obvious cytotoxicity of BCL-NEs was observed. After 24 hours of incubation, the survival rate of all cells remained >90%, indicating the low cytotoxicity of BCL-NEs. In the NE system we developed, PM and sodium oleate are the preferred surfactants. They are all low-toxic and degradable in the body, so they will not cause obvious cytotoxicity. Emulsion is a popular dosage form that patients are willing to take. Low toxicity and compliance endow BCL-NEs with vigor as a potential oral delivery vehicle.

Figure 7 Relative cell viability of Caco-2 incubated with nanoemulsions with different BCL levels (n=3, mean ± StD). Abbreviations: BCL, baicalein; StD, standard deviation.

In this article, a new NE formulation based on hemp oil and less surfactant was developed for oral administration of BCL. The applicability of NE as an oral delivery vehicle for BCL was evaluated. NEs are easily produced using HPH technology and have a small particle size (~90 nm). High BCL interception and low BCL release are realized through NE. In vivo pharmacokinetics showed that NEs significantly increased the oral bioavailability of BCL. The improvement in oral absorption of BCL can be attributed to the promotion of transcellular transport due to the encapsulation in NEs. In addition, the NE we developed has perfect biocompatibility due to the use of less surfactants. This research provides valuable information for designing innovative NE to more effectively deliver malabsorbed drugs through the oral route.

This study was funded by the Key Research Project of the Department of Science and Technology of Henan Province (No. 112102310306). Thanks to the Department of Pharmacy, Huaihe Hospital, Henan University.

The authors report no conflicts of interest in this work.

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